Tooth organogenesis is dependent upon a series of reciprocal-, instructive signals. These signals culminate in the induced-, regulated expression of the amelogenin gene. Amelogenin is the hallmark of tooth development and its expression is dependent on numerous instructive signals. We have shown that expression of the mouse amelogenin gene is tightly coupled to a repressive pathway mediated through Msx2 and an activation pathway mediated by C/EBPalfa. Msx2 opposes the C/EBPalpha-mediated activation by directly interacting with C/EBPalfa at the protein level. In mice homozygous for inactivation of the Msx 2 gene, secretory ameloblast function is largely normal. However, in LS-8 cells, an ameloblast-like cell line, the suppressive effect of Msx2 on the amelogenin promoter can be overcome by increasing the levels of ectopic C/EBPalfa. Taken together, these findings suggest that Msx2 plays a less critical role in transcriptional activation of the amelogenin gene than does C/EBPalfa. With the critical role for C/EBPalfa in regulating amelogenin gene expression, we will focus attention on the cellular and molecular biology of C/EBPalfa expression in developing amelpblasts and ameloblast-like cell lines. In specific aim 1, we will test the hypothesis that expression of C/EBPalfa by ameloblasts is dependent upon the expression of family members C/EBP-beta and C/EBP-delta to activate C/EBPalfa expression. In Specific Aim 2 we will test the hypothesis that the phosphorylation status of full length CIEBPalfa is critical to activation. In Specific Aim 3 we will identify transcriptional coalfactivators that interact with C/EBPalfa In Specific Aim 4, we will use the BMP-5 signaling pathway to induce C/EBPdelta in non -ameloblasts of the lingual inner enamel epithelium thereby activating amelogenin gene expression. The long-term biomedical goal of this application is to better understand the regulated induction of amelogenin gene transcription in developing mouse teeth by better understanding the C/EBPdelta activation pathway. Armed with such knowledge, we may be able to design strategies to re-activate amelogenin expression as part of a long-term strategy to replace human dental enamel tissue lost to disease or congenital defect.